Thrombin (α-thrombin) is an endolytic serine protease that plays a major role in the coagulation cascade or thrombus formation. Thrombin is produced by the enzymatic cleavage of the zymogen prothrombin. The cleavage is carried out by activated factor X (factor Xa). Human thrombin is composed of a light chain (having a molecular weight of ˜6 kDa) and a heavy chain (having a molecular weight of ˜31 kDa). Thrombin comprises an active site which is located within the heavy chain. Thrombin has a high specificity for protein substrates having arginine bonds and its main substrate is fibrinogen. Thrombin binds fibrinogen and converts it into fibrin. Thrombin also activates protein C, platelets, factor XIII and plasma procarboxypeptidase B (TAFI).
Normally, thrombin activity is inhibited in plasma. The main inhibitor of thrombin present in plasma is anti-thrombin III (AT-III) and to a lesser extent heparin cofactor II. The rate of inhibition by both of these inhibitors is profoundly increased in the presence of optimal concentrations of heparin. Other physiological inhibitors of thrombin are α2-macroglobulin, and α1-antitrypsin1-4.
AT-III inhibits the coagulation cascade by binding thrombin and factor Xa. The inhibitory activity of AT-III is markedly increased by the presence of heparin. The normal anti-thrombin III concentration in human blood plasma is high and is of approximately 0.12 mg/ml. Thrombin inactivation occurs as a result of trapping of thrombin in an equimolar complex with AT-III. Formation of the complex prevents accessibility of the active site of the thrombin to its substrate. Formation of an anti-thrombin III-thrombin complex involves an interaction between the thrombin and a specific reactive peptide bond within AT-III. In human AT-III this bond is between arginine (arg) 393 and serine (ser) 394.
Abnormal high levels of thrombin in the circulation may trigger the coagulation cascade, consequently resulting in generalized coagulopathy. A method which enables fast and quantitative determination of the levels of thrombin in a tested biological sample, e.g. a blood sample, is of considerable biomedical interest. Fast and quantitative analysis of thrombin in plasma of humans and animal models may also be of high importance for safety assessments of experimental and routine usage of hemostats which include thrombin.
Detection of thrombin by immunoassay using thrombin specific antibodies, in a background of relatively large quantities of the pro-enzyme prothrombin, such as in a plasma sample, may be challenging.
U.S. Pat. No. 4,753,875 discloses a method for assaying thrombin, in which an excess of tagged thrombin inhibitor (e.g. tagged hirudin or tagged AT-III) is mixed in solution with a tested sample containing the thrombin to form a mixture of a tagged inhibitor-thrombin complex and free tagged inhibitor. Next, an anti-thrombin antibody is added to form complexes of tagged inhibitor-thrombin and thrombin specific antibody. The complexes are then separated from the free tagged inhibitor and the tag is measured. Exemplified is an assay for testing thrombin in a sample of plasma by adding 3H-hirudin and separating the complex thrombin-hirudin with thrombin specific antibodies coupled to cellulose. Thus, this test requires a step of complex separation e.g. using an additional step of affinity chromatography column separation.
U.S. Pat. No. 5,296,352 discloses monoclonal antibodies which are directed against and recognize the thrombin/hirudin-complex and derivatives thereof. U.S. Pat. No. 5,296,352 discloses the use of the monoclonal antibodies for detection of thrombin/hirudin complex. It is also disclosed that the monoclonal antibodies can be employed for detecting small amounts of spontaneously generated thrombin in a subject. The method includes injecting to the subject hirudin as a tracer followed by the measurement of the complex thrombin/hirudin formed with the aid of the monoclonal antibodies that recognize the complex. Exemplified is an ELISA assay for determination of the thrombin-hirudin complex present in plasma. This test requires injection of hirudin to the subject for testing thrombin.
There is a need for an in-vitro, easy, fast, and effective test for determining thrombin levels in a blood sample.